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Nanomaterials promise to improve disease diagnosis and treatment by enhancing the delivery of drugs, genes, biomolecules and imaging agents to specific subcellular targets. In order to optimize nanomaterial design for this purpose, a comprehensive understanding of how these materials are taken up and transported within the cell is required. In this review, we discuss the endocytic pathways employed by different types of nanoparticles with emphasis on the influence of nanoparticle surface modification. The use of pharmacological inhibition to probe internalization and intracellular trafficking pathways of nanoparticles is critically evaluated. Finally, approaches to target-specific delivery of therapeutics via nanoparticles into the cytoplasm and nucleus are addressed.